CN107022495A - Two plants thick wall spore Pu Keniya bacterium fungi and its application - Google Patents
Two plants thick wall spore Pu Keniya bacterium fungi and its application Download PDFInfo
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- CN107022495A CN107022495A CN201710265538.9A CN201710265538A CN107022495A CN 107022495 A CN107022495 A CN 107022495A CN 201710265538 A CN201710265538 A CN 201710265538A CN 107022495 A CN107022495 A CN 107022495A
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Abstract
The present invention relates to two plants thick wall spore Pu Keniya bacterium fungi and its application, belong to microorganism fungus kind technical field.Thick wall spore Pu Keniya bacterium Pochonia chlamydosporia YMF1.00615 and thick wall spore Pu Keniya bacterium Pochonia chlamydosporia YMF1.00111, preserving number is respectively CGMCC No.9586 and CGMCCNo.9585.Thick wall spore Pu Keniya bacterium Pochonia chlamydosporia YMF1.00615 and thickness wall spore Pu Keniya bacterium Pochonia chlamydosporia YMF1.00111 are preparing anti-nematode active material aurovertin (aurovertins) and the application in anti-root-knot nematode.The present invention has following beneficial effect to be：Contain the anti-nematode active material aurovertin for reaching 800 1500 μ g/mL contents in PDB zymotic fluids in thick wall spore Pu Keniya bacterium Pochonia chlamydosporia YMF1.00615 and thickness wall spore Pu Keniya bacterium Pochonia chlamydosporia YMF1.00111, with very strong malicious eelworm-killing activity, it can be applied in poisoning plant pathogeny line insect preparation is prepared.
Description
Technical field
The present invention relates to two plants thick wall spore Pu Keniya bacterium fungi and its application, belong to microorganism fungus kind technical field.
Background technology
Plant pathogeny line insect is a kind of to endanger serious plant disease, it was reported that plant pathogeny line insect belong to up to more than 200
More than 5000 plant.Plant pathogeny line insect disease brings nearly 125,000,000,000 dollars of economic loss to whole world agricultural every year, is only second to true
Fungus diseases.Biocontrol of nematodes is attracted attention, and national governments also give very big support, and first generation nematode biology is anti-
The live bacteria agent such as nematode-trapping fungi, inner parasitic epiphyte and parasitics bacterium for controlling agent arise at the historic moment, but it is by such environmental effects
Greatly, less stable, the problems such as shelf life is short, be restricted its application.To overcome this disadvantage, domestic and international researcher is more
Concern second generation nematode bio-control factors are Metabolite research and development.The secondary metabolite of fungi is used as second generation nematode
Bio-control factors, are played a role in the way of directly acting on crops nematode, have that effect is fast, drug effect is high, Environmental compatibility is good,
The advantages of cannot be easily caused secondary pollution.Early-stage Study is we have found that fungal bacterial strain can be by producing aurovertin
(aurovertins) class secondary metabolite is used as virulence factor kill nematode, therefore finds and screen and can efficiently produce
The fungal bacterial strain of aurovertin class compound, lays the foundation for the research and development that prepare efficient nematicidal toxicity fungi preparation.
The content of the invention
It is an object of the invention to provide two plants thick wall spore Pu Keniya bacterium fungi and its application.
The purpose of the present invention is achieved through the following technical solutions:
The thick wall spore Pu Keniya bacterium Pochonia chlamydosporia YMF1.00615 of separation screening of the present invention and
Thick wall spore Pu Keniya bacterium Pochonia chlamydosporia YMF1.00111, on 09 04th, 2014 is deposited in
State's Microbiological Culture Collection administration committee common micro-organisms center；Depositary institution address：BeiChen West Road, Chaoyang District, BeiJing City 1
No. 3 Institute of Microorganism, Academia Sinica of institute；Preserving number is respectively CGMCC No.9586 and CGMCC No.9585.
The thick wall spore Pu Keniya bacterium Pochonia chlamydosporia YMF1.00615 of separation screening of the present invention and
Thick wall spore Pu Keniya bacterium Pochonia chlamydosporia YMF1.00111 are preparing anti-nematode active material
Application in aurovertins and anti-plant pathogeny line insect preparation.
Compared with prior art, the present invention has following beneficial effect to be：
Thick wall spore Pu Keniya bacterium Pochonia chlamydosporia YMF1.00615 and thick wall spore Pu Keniya bacterium
Contain in PDB zymotic fluids in Pochonia chlamydosporia YMF1.00111 and reach 800-1500 μ g/mL contents
Anti- nematode active material aurovertin, with very strong malicious eelworm-killing activity, can kill plant pathogeny line insect preparation preparing
Middle application.
Brief description of the drawings
Fig. 1 shows TLC detections Pochonia chlamydosporia zymotic fluid ethyl acetate portions (C) and mycelium methanol
The result of crude extract (M).Wherein：Compound 1：Aureomycin I (aurovertin I), compound 2：Aureomycin E (aurovertin
E), compound 3：Aureomycin F (aurovertin F), compound 4：Aureomycin D (aurovertin D).
Fig. 2 is the structure of compound 1-4.
Fig. 3 be HPLC detect two fungal strain Pochonia chlamydosporia YMF1.00615 and YMF1.00111 with
The comparing result for the YMF1.00613 zymotic fluid aurovertin class contents reported.Abscissa is the time, and ordinate is absworption peak
Abundance, bacterial strain YMF1.00615 and YMF1.00111 aureomycin D absworption peak abundance reaches 400mAU, has reported
YMF1.00613 absworption peak abundance is then 250mAU.
Embodiment
Embodiment 1
(1) bacterial strain Pochonia chlamydosporia YMF1.00615 and YMF1.00111 culture：
YMF1.00615 in PDA solid mediums and YMF1.00111 is distinguished into Conventional activation 3 days, in aseptic condition
Under, by 2x2cm2The fungus block of size is inoculated in the 500mL triangular flasks equipped with 250mL PDB culture mediums, 28 DEG C, 160rmin-1
Shaking table culture 7 days.
PDA solid mediums are：Peeled potatoes 200g, boils 30min, 4 layers of filtered through gauze, in filtrate after being cut into small pieces
Middle addition 20g glucose, adds water and is settled to 1L, add 1.5% (m/v) agar, 121 DEG C sterilizing 20 minutes after it is stand-by；
PDB fluid nutrient mediums：Peeled potatoes 200g, is cut into after 1x 1x 1cm fritters and boils 30min, 4 layers of gauze mistake
Filter, in filtrate add 20g glucose, add water and be settled to 1L, 121 DEG C sterilizing 20 minutes after it is stand-by.
Embodiment 2 bacterial strain Pochonia chlamydosporia YMF1.00615 and YMF1.00111 zymotic fluid is carried
Take and TLC detections
The strain fermentating liquid of above-mentioned implementation 1 is filtered, filtrate is after fermenation raw liquid is concentrated under reduced pressure, to use isometric ethyl acetate
Extraction three times, is then combined with ethyl acetate evaporated under reduced pressure.Mycelium ethanol or acetone soak are after 12 hours, by methanol extract liquid
Evaporated in vacuo.Distinguish soaking fermentation liquid ethyl acetate portion and mycelium methanol crude extract with 100mL acetone, ultrasonic wave 20min,
The detection of thin-layer chromatography TLC plates is carried out, with chloroform：Methanol=15:1 be panel system when, UV detect when show yellowish green under 365nm
Color fluorescence, shows blackening, 10%H under 254nm2SO4It is purple when alcoholic solution sprays and heats colour developing, detects several phases
As band (See Figure 1), wherein the content highest of (M in Fig. 1) compound 4 in mycelium methanolic extract, in zymotic fluid
The content highest of ethyl acetate portion (C in Fig. 1) compound 3.Four kinds of Aurovertin metabolins compound 1-4 structure is as schemed
Shown in 2.
The gold of embodiment 3 bacterial strain Pochonia chlamydosporia YMF1.00615 and YMF1.00111 zymotic fluid
Vertimycin aurovertins content analysis
HPLC detects that sample preparation methods are：1 PDB zymotic fluids will be implemented, through 0.22 μm of membrane filtration and load HPLC
Sample bottle, be put in 4 DEG C it is standby.Using high performance liquid chromatograph HP 1200unit (Agilent, Waldbronn, Germany),
It is equipped with CAPCELL PAK C18,5 μm, 4.6 × 250mm (Shiseido) reversed-phase column.Mobile phase is trifluoroacetic acid aqueous solution and deionization
Water (the acetonitrile of 10% acetonitrile -95%.Elution flow rate is 1mL/min.40 DEG C of column temperature, the μ L of applied sample amount 25.With aurovertin
(aurovertins) D, E, I, F are aurovertin content in control, detection zymotic fluid, and as shown in table 1, UV is detected eluent system
Wave band is 220-400nm.
The eluent system of aurovertin in the thick wall spore Pu Keniya bacterium PDB zymotic fluids of table 1.HPLC detections
As shown in fig. 3, it was found that with reported production aurovertin bacterial strain Pochonia chlamydosporia
YMF1.00613 is compared, and contains higher amount in the PDB zymotic fluids of two plants of bacterial strain YMF1.00615 and 1.00111 in the present invention
Anti- nematode active material aurovertin, content is beyond will by about one time, and total content reaches 800-1500 μ g/mL.Wherein
The proportional amount highest of aurovertin D in YMF1.00615.
Embodiment 4 bacterial strain Pochonia chlamydosporia YMF1.00615 and YMF1.00111 zymotic fluid is killed
The test of nematode ability
The mycelium of embodiment 1 is filtered：Bacterium solution is separated with mycelium using 0.22 μm of filter membrane, by bacterium solution at 120 DEG C
Sterilize 20min.With the eelworm-killing activity of liquid immersion method test strain zymotic fluid：Root-knot nematode Meloidogyne
Incognita (race 3) cultures (25 DEG C) tomato root in greenhouse.Tomato diseased plant of the collection with root-knot nematode pieces of an egg, will
Old complaint cuts into about 2cm segments, with the ripe pieces of an egg of the dissecting needle picking of sterilizing and is placed on 40 μm of cell screen clothes.By screen cloth and
Pieces of an egg are placed in 1min in 2% liquor natrii hypochloritis, during which weak vibrations screen cloth.Screen cloth is put after aseptic water washing pieces of an egg 5-7 times
It is placed in the 6cm culture dishes equipped with 15mL sterilized waters hatching in 28 DEG C of constant incubators and obtains second instar larvae (J2) use in 24 hours
In toxotest.
Using 24 porocyte culture plates, 1mL ferment filtrates and 10 μ L nematodes suspensions (average 500 lines are added in every hole
The μ L of worm/10), after 20 DEG C (C.elegans) or 28 DEG C (M.incognita) stand 24 hours, nematode is counted under the microscope dead
Die rate.Identify that the dead method of nematode is that dead nematode is stiff or in " J " type, and living nematode then crimps twisting.Stir quiet with needle point
Only each portion of nematode body, nematode does not make any reaction and is then accredited as death yet.
The death rate=(verge of death borer population/(verge of death borer population+hot line borer population)) × 100%
Corrected mortality=test sample the death rate-control group death rate
Using the PDB fluid nutrient mediums of non-inoculated fungi as control, whole experiment in triplicate, takes three average values, calculates
Go out average mortality and corrected mortality.
Experimental result is as follows：
The 24 hours Toxic test results of strains tested fermentation liquor treatment nematode of table 2
As shown in table 2, bacterial strain Pochonia chlamydosporia YMF1.00615 and YMF1.00111 zymotic fluid
The PDB fermentation liquor treatment plant nematodes M.incognita of bacterial strain is after 24 hours, and the death rate of nematode is above 95%, says
The zymotic fluid of bright strain bacterium shows very strong malicious eelworm-killing activity.
Claims (3)
1. two plants thick wall spore Pu Keniya bacterium fungies are respectively thick wall spore Pu Keniya bacterium Pochonia chlamydosporia
YMF1.00615 and thick wall spore Pu Keniya bacterium Pochonia chlamydosporia YMF1.00111, preserving number is respectively
CGMCC No.9586 and CGMCCNo.9585.
2. preserving number is CGMCC No.9586 thick wall spore Pu Keniya bacterium Pochonia chlamydosporia
Applications of the YMF1.00615 in anti-nematode active material aurovertins and anti-plant pathogeny line insect preparation is prepared.
3. preserving number is CGMCC No.9585 thick wall spore Pu Keniya bacterium Pochonia chlamydosporia
Applications of the YMF1.00111 in anti-nematode active material aurovertins and anti-plant pathogeny line insect preparation is prepared.
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| CN201710265538.9A CN107022495B (en) | 2017-04-21 | 2017-04-21 | Two strains of Pochonia chlamydosporia fungus and application thereof |
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| CN201710265538.9A CN107022495B (en) | 2017-04-21 | 2017-04-21 | Two strains of Pochonia chlamydosporia fungus and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107955802A (en) * | 2017-12-05 | 2018-04-24 | 福建农林大学 | A kind of method of thickness wall spore Pu Keniya bacteria solid fermentations production spore |
| CN112063536A (en) * | 2020-09-04 | 2020-12-11 | 青岛和协生物科技有限公司 | Pochonia chlamydosporia for preventing and treating root knot nematode disease, composite powder and application |
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|---|---|---|---|---|
| CN1513975A (en) * | 2003-06-03 | 2004-07-21 | 云南大学 | Method of producing large quantity thick wall spore by thick wall spore verticillium liquid fermentation |
| CN104560723A (en) * | 2014-09-05 | 2015-04-29 | 云南大学 | Pochonia chlamydosporia strain and screening and application thereof |
-
2017
- 2017-04-21 CN CN201710265538.9A patent/CN107022495B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1513975A (en) * | 2003-06-03 | 2004-07-21 | 云南大学 | Method of producing large quantity thick wall spore by thick wall spore verticillium liquid fermentation |
| CN104560723A (en) * | 2014-09-05 | 2015-04-29 | 云南大学 | Pochonia chlamydosporia strain and screening and application thereof |
Non-Patent Citations (1)
| Title |
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| 王彦利: "厚垣孢普克尼亚菌产生的金轮霉素对秀丽隐杆线虫作用靶点的研究", 《中国博士学位论文全文数据库_农业科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107955802A (en) * | 2017-12-05 | 2018-04-24 | 福建农林大学 | A kind of method of thickness wall spore Pu Keniya bacteria solid fermentations production spore |
| CN112063536A (en) * | 2020-09-04 | 2020-12-11 | 青岛和协生物科技有限公司 | Pochonia chlamydosporia for preventing and treating root knot nematode disease, composite powder and application |
| CN112063536B (en) * | 2020-09-04 | 2022-07-05 | 青岛和协生物科技有限公司 | Pochonia chlamydosporia for preventing and treating root knot nematode disease, composite powder and application |
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